PROJECT 4 - ABSTRACT Systemic Lupus Erythematosus (SLE), is the prototype of human autoimmune disease mediated by autoantibodies. In contrast to antibodies directed against dsDNA which fluctuate with disease activity and treatment, highly pathogenic autoantibodies against RNA-binding proteins (RBP), are present years before disease onset and persist in very high titers throughout disease irrespective of disease activity and treatment, including B cell depletion. These features support the notion that RBP antibodies are produced by plasma cells (PC) of prolonged longevity that don't require replenishment from B cell precursors. However, this concept and multiple other aspects of PC biology and its subversion in human autoimmunity in general and SLE in particular remain poorly understood. These limitations are created to a large extent by the lack of definition of different PC populations and a precise identification of the cellular phenotype of human LLPC. Our laboratory has recently provided the first characterization of the diversity and origin of peripheral PC in active SLE and in collaboration with Dr. Lee (Project 3) has also provided the first description of LLPC in the human BM. These studies and additional preliminary results suggest that SLE PC are characterized by prolonged survival even at an early stage of differentiation. In all, we hypothesize that SLE is characterized by increased generation of PC with prolonged survival and that these feature lead to the accumulation of autoreactive LLPC in the SLE BM. We further hypothesize that pathogenic RBP LLPC accumulate through a process of ongoing generation from precursor B cells, sequential differentiation through consecutive maturation stages and prolonged survival within the BM microenvironment. The generation and survival of SLE PC will be tested using state-of-the-art methods for deep sequencing of PC repertoires; ultra-high throughput single cell PC antibody interrogation; in vivo labeling with heavy glucose; and in vitro PC cultures supported by innovative BM microenvironments developed by Dr. Lee in Project 3. Studies will be performed with PC generated during the natural course of SLE and in response to patient vaccination. Finally, through Core B, we will provide a comprehensive definition of the molecular programs responsible for the behavior of SLE PC using integrated transcriptional and epigenetic studies. The knowledge generated by this project will be invaluable to understand the pathogenic role and mechanisms of PC in SLE and to design better and safer therapeutic strategies for this disease. Specifically, establishing the continued production of RBP PC would indicate the need for combined targeting of PC and their B cell precursors. Our results will also identify PC biomarkers to monitor disease development, segment patients for personalized treatment and monitor the outcome of therapeutic interventions.